Exploring protein-protein interactions by Weak Affinity Chromatography (WAC) - IL-23 case study

This is a presentation of the method of weak affinity chromatography and its application in a case study. The presentation was held at the Drug Discovery Chemistry meeting in San Diego on April 2-4, 2024, by Björn Walse, CEO of SARomics Biostructures.

1. Exploring protein-protein interactions by Weak
Affinity Chromatography (WAC™)An IL-23 case study
Björn Walse, CEO, SARomicsBiostructures, Lund, Sweden
Drug Discovery Chemistry, April 2-4, 2024

2. Our FBLD platform
55 employees
•Medicinal chemistry
•Computational chemistry
•Peptide chemistry
•Analytical chemistry
•In vitro ADME
•In vitro biology
28 employees
•X-ray crystallography
•NMR
•SBDD
•Computational chemistry
•Biophysics
•Protein chemistry
FBLD Projects
•Target ligandability
•Ranking of XFS hits
•Fragment screening
•Active-to-Hit and Hit-to-Lead campaigns
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FBLD Technologies
•WAC™
•NMR
•Thermal shift assay (TSA)
•X-ray crystallography
•In silico screening
•Fragment libraries (250 or 550 cmpds)
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3. Quick intro to WAC™
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•Affinity chromatography with immobilized target (protein load –2-5mg)
•MS-detection enables screening at low μM, built-in QC
•Affinity range low μM to mM, direct detection with immediate KDranking
•High throughput (>5000 cmpds/week; cocktails of 25-100)
•Used along TSA, NMR, X-ray for integrated hit finding, validation and progression workflow

4. Working with WAC for 7 years
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Facts and learnings
•> 50 FBLD projects over 7 years
•20 distinct target classes
•WAC hit rates from 1% to 20%, avg 6%
•Library quality and size matters
•Best follow-up: NMR, SPR, X-ray
Quirks, drawbacks
•Buffer with low ionic strength, less charge shielding
•Ranking order can be disrupted by ionic interactions
•Non-volatile additives not tolerated by MS
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3333
222
1111111111
kinaseGTPase
cytokine/PPIbromodomaindeubiquitinase
integrin
helicase/ATPasesignalling proteintranscription factor
other receptorsautophagy factor
deaminaseesterasegalectinligase
lipoxygenase
synthasetransferase
ubiquitin ligase
viral methyl transferase
Target class occurrence

5. Targeting IL-23
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•Interleukin 23(IL-23) is aheterodimericinflammatory cytokinecomposed of anIL-
12B(p40) and anIL-23A(p19) subunit
•IL-23 signals through a receptor complex formed by IL-12Rb1 and IL-23R
•Key cytokine forT helper type 17 cell(Th17 cell) maintenance and expansion
•Th17 cells and IL-23 signaling play an important role in the immune response against
extracellular pathogens
•Aberrant Th17 isassociated with multiple autoimmune conditions
•Clinically, antagonist antibodies targeting IL-23 (ustekinumab) have been approved
for the treatment of moderate to severe plaque psoriasis, psoriatic arthritis, Crohn’s
disease, and ulcerative colitis
•An orally administered small-molecule inhibitor of the IL-23 pathway has the
potential to provide significant benefitsfor patients suffering from autoimmune
inflammatory disorders
•Identifying small molecule inhibitors of the IL-23 pathway has proven to be a
challenging process
IL-23: p19/p40 complex
PDB code: 3DUH
Luparduset. al., 2008, JMB, 382, 931-941.

6. Exploring IL-23 by WAC™
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Fragment screening
Hit validation
Binding site validation
X-ray crystallography
Set-up / Feasibility

7. Exploring IL-23: Feasibility
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Fragment screening
Hit validation
Binding site validation
X-ray crystallography
Set-up / Feasibility•IL-23 (6mg) was immobilized using reductive amination between
the aldehyde groups on the silica surface and the lysine groups of
the protein
•Remaining aldehyde groups were reacted with ethanolamine
using reductive amination
•The silica with the protein was packed into an empty LC-column

8. -4
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∆RT (min)
Exploring IL-23: WAC screening
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•WAC screenwith fragment library
•1013 cmpds
•Diverse set
•Screened at 1 µM
•19 hits (2% hit rate)
•Top WAC hit ΔRT = 10.5 min
•Hit threshold = 0.5 min
Fragment screening
Hit validation
Binding site validation
X-ray crystallography
Set-up / Feasibility
ΔRT 0.8 min
RG200616
ΔRT 10.5 min
RG200734

9. Hit validation: NMR and TSA
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•Ligand detected NMR (T1ρ-experiment)
•12 out of 19 WAC hits tested
•6 cmpdsvalidated -> nominated for TSA
•Thermal shift assay (TSA)
•6 NMR hits + 5 other WAC hits tested
•Tm: 1.4 –2.2°C stabilizing (2 cmpds)
•Tm: 2.1 –7.8°C destabilizing (7 cmpds)
•One cmpdhigh initial fluorescence
•One cmpdno effect
•6 cmpdsnominated for X-ray
Fragment screening
Hit validation
Binding site validation
X-ray crystallography
Set-up / Feasibility
1.781.801.821.841.862.162.182.202.222.242.262.402.422.442.462.482.502.742.762.782.802.882.902.922.942.962.983.003.023.04
f1 (ppm)
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190506_B9.14.fid
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10. Hit validation: X-ray crystallography
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RG200734
WAC ΔRT:10.5 min
Mw:216 Da
Resolution:2.70 Å
RG200616
WAC ΔRT:0.8 min
Mw:188 Da
Resolution:2.95 Å
Fragment screening
Hit validation
Binding site validation
X-ray crystallography
Set-up / Feasibility
IL-23p19p40
D1
D2
D3

11. Hit validation: X-ray crystallography
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RG200734
WAC ΔRT:10.5 min
Mw:216 Da
Resolution:2.70 Å
RG200616
WAC ΔRT:0.8 min
Mw:188 Da
Resolution:2.95 Å
Fragment screening
Hit validation
Binding site validation
X-ray crystallography
Set-up / Feasibility
Trp143
Arg139
Lys234
Lys134
Glu132
Glu188
Ser176
Thr141
Ser190

12. Binding site validation
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IL-12Rb1
IL-23p19p40
IL-23R
D1
D2
D3
D1
D2
D1
D2
D3
Structures of complete extracellular receptor assemblies mediated by IL-12 and IL-23
Bloch et al., 2024, Nat Struct Mol Biol, January 29.
PDB code: 8OE4
=> No blocking of receptor interaction!
D3
D4
D5

13. Blocking PPIs
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PPI fragments:
•Appear to be heavier and more
lipophilic
•Higher occurrence of acid-and
base-containing functional groups
•Higher proportion of hydrophobic
atoms
Applied Biophysics for Drug Discovery, 2017
Eds. Donald Huddler, Edward R. Zartler
Chapter 7
StenOhlsonand Minh-Dao Duong-Thi
Weak Affinity Chromatography (WAC)
Chapter 3
BjörnWalse, Andrew P. Turnbull and Susan M. Boyd
Tailoring Hit Identification and Qualification Methods
for Targeting Protein–Protein Interactions
Walse, Turnbull & Boyd (2017) In Applied Biophysics for Drug Discovery,pp. 29-59.
Turnbull, Boyd & Walse (2014) Res. Rep. Biochem. 4, 13-26.
Comparison of physicochemical and shape properties
of 100 fragments active against PPI targets versus
100 fragments active against non-PPI targets

14. Is RG200734 a “general” PPI binder?
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Properties
•Lipophilic with high pKa
•Two hydrogen bond acceptors
•Higher proportion of hydrophobic
atoms
RG200734 is a hit in WAC screens towards several different
targets with overweight on PPI targets
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IL23USP7SMARCA4Target ATarget BTarget CTarget DTarget ETargetFTarget GTarget HTarget ITarget JTarget KTarget LTarget MTarget NTarget OTarget PTarget QTarget RTarget STarget TTarget U
∆RT
(min)

15. Conclusions and acknowledgement
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Visit us @MediconVillage, Lund, Sweden
or
pop by our posters:
P080: Two Approaches Aiming to Prevent Phosphorylation of
SMARCA4 by CDK9 Using WAC and In Silico Screening,presented
by Johan Evenäs, Red GleadDiscovery AB
P085: Fragment Screening of IL-23 by Weak Affinity
Chromatography (WAC),presented by KenthHallberg, SARomics
Biostructures AB
Martin Welin
Carl Diehl
Masato Akutsu
Raymond Kimbung
Nadia Rose
Bo Svensson
Maria Håkansson
Kenth Hallberg
Björn Walse
•WACscreenrevealsanumberofIL-23hits,validatedbyNMRandTSA
•X-raycrystalstructuresidentifiesuniquebindingsite
•Difficulttoprogresshitsduetolackofrelevantbiologicalactivity
•Subsequent published structures of IL-23 receptor complex de-
validates binding site
•PPI-likefragmenthits
Kirill Popov
Stella Timpka
Henrik von Wachenfeldt
Jessica Larsson
Johan Evenäs
www.saromics.comwww.redglead.com
Medicon Valley life science cluster in
the Greater Copenhagen region